PROJECT SUMMARY The formation of bone in the mandible requires that neural crest mesenchyme (NCM) undergoes an inductive interaction with adjacent epithelium. This interaction relies on a number of yet to be identified signals that permit the NCM to differentiate into bone. Thus, identifying molecular signals mediating this epithelial-mesenchymal interaction (EMI) would allow us to discover proteins that regulate mandibular osteogenesis, and which potentially could be used therapeutically to generate bone in cases of disease and injury. One signaling molecule currently known to participate in these EMI and promote osteogenesis in the jaw is BMP4. However, BMP4 alone is not sufficient to generate bone in mandibular mesenchyme cultured without adjacent epithelium. The overall goal of this project is to identify other osteoinductive signals and test if they can be used in combination with BMP4 to function in place of the epithelium that normally induces bone in the jaw. An RNA-seq experiment and preliminary data reveal that spatiotemporal changes in several candidate genes including members of the WNT and CXC signaling pathways are present at the right time, place, and levels to mediate the osteogenic EMI in the mandible. This project will investigate the role of these genes and test the extent to which they govern the EMI required for bone formation in the mandible. The strategy employs a unique avian chimeric system and involves transplanting NCM, which generates all of the osteogenic precursor cells in the mandible between quail and duck embryos. Aim 1 will characterize the spatiotemporal expression of these genes qualitatively and quantitatively on the mRNA and protein levels, and test if their expression is mediated by NCM. Aim 2 will use in vitro organ culture and tissue recombinations to test if expression of these genes in mandibular mesenchyme requires epithelial signaling. Aim 3 will employ gain- and loss-of-function approaches to identify the role of these genes and test if these genes regulate the timing of mandibular osteogenesis and if can they induce bone when administered either alone or in various combinations with BMP4 in the absence of mandibular epithelium. This project will lead to the discovery of in vivo mechanisms through which NCM controls bone formation in the jaw skeleton and has clinical relevance by testing the efficacy of specific molecules that could ultimately be used therapeutically to benefit human patients. Completion of this fellowship will provide the scientific and technical training necessary for advancing the career of a dentist-scientist studying craniofacial development.